The multiple antibiotic resistance (mar) operon of the enteric bacterium Escherichia coli regulates multiple antibiotic resistance and pathogenicity. The goal of the present study is to improve our understanding of how the Mar system achieves its effects. The marRAB operon encodes MarR, a protein whose sole known function is to repress the expression of its own operon and which is inactivated by small inducer molecules (ligands); MarA, a master regulatory protein; and MarB, which negatively affects marA expression. Aim A of the study will characterize how ligands inactivate MarR, and how the putatively periplasmic MarB protein represses marA expression. Aim A also involves novel observations on the multidrug efflux pump AcrAB-TolC, which is up-regulated by MarA. Inactivation of the AcrAB-TolC pump gives rise to enhanced expression of the genes for AcrAB and for MarA, suggesting that cellular metabolites, normally exported by the pump, may be inducers of acrAB and marRAB. Genetic inactivation of different metabolic pathways led us to identify several metabolites potentially responsible for such effects; their mechanisms of action will be studied. Additional inducing metabolites will also be identified by a variety of techniques including metabolomics. Substrate antibiotics also induce the expression of the AcrAB-TolC pump; the underlying mechanism will be studied. Aim B investigates the small RNAs (sRNAs) regulated by MarA and by its two homologs SoxS and Rob, which together with MarA, regulate the transcription of at least forty protein-specifying genes directly. Although expression of two sRNAs (MicF and CsrA) has been shown to be regulated by Mar/Sox/Rob, there has of yet been no systematic search to identify similarly- regulated sRNAs. This search will use innovations in transcriptomic technologies. Aim C arises from observations that MarA/SoxS/Rob enhance the ability of bacteria to persist in the murine kidney in a model of ascending pyelonephritis. Aim C will focus on the bacterial targets and pathways involved, including that of the carbon storage regulation system (already an identified target of Mar/Sox/Rob), as well as on the host response to the infection. Strains with and without the genes encoding these three regulators will be compared using in vitro cell culture and in vivo histopathology to assess differential expression patterns of the host immune response. Bacterial pathogenic factors such as those affecting attachment and invasion will also be studied. In summary, this proposal investigates the Mar system and its homologs SoxS/Rob of E. coli, which control many genes involved in antibiotic resistance and pathogenicity. A deeper knowledge of this system will contribute to knowing how this bacterium, and other bacteria with homologous regulators, resist antibiotics and host defenses, and thus will provide leads for improved therapeutic approaches.